Respiratory complex I transfers electrons from NADH to quinone, utilizing the reaction energy to translocate protons across the membrane. It is a key enzyme of the respiratory chain of many prokaryotic and most eukaryotic organisms. The reversible NADH oxidation reaction is facilitated in complex I by non-covalently bound flavin mononucleotide (FMN). Here we report that the catalytic activity of E. coli complex I with artificial electron acceptors potassium ferricyanide (FeCy) and hexaamineruthenium (HAR) is significantly inhibited in the enzyme pre-reduced by NADH. Further, we demonstrate that the inhibition is caused by reversible dissociation of FMN. The binding constant (Kd) for FMN increases from the femto- or picomolar range in oxidized complex I to the nanomolar range in the NADH reduced enzyme, with an FMN dissociation time constant of ~ 5 s. The oxidation state of complex I, rather than that of FMN, proved critical to the dissociation. Such dissociation is not observed with the T. thermophilus enzyme and our analysis suggests that the difference may be due to the unusually high redox potential of Fe-S cluster N1a in E. coli. It is possible that the enzyme attenuates ROS production in vivo by releasing FMN under highly reducing conditions.